Human osteoarthritis knee joint synovial fluids cleave and activate Proteinase-Activated Receptor (PAR) mediated signaling

Osteoarthritis (OA) is the most prevalent joint disorder with increasing worldwide incidence. Mechanistic insights into OA pathophysiology are evolving and there are currently no disease-modifying OA drugs. An increase in protease activity is linked to progressive degradation of the cartilage in OA. Proteases also trigger inflammation through a family of G protein-coupled receptors (GPCRs) called the Proteinase-Activated Receptors (PARs). PAR signaling can trigger pro-inflammatory responses and targeting PARs is proposed as a therapeutic approach in OA. Several enzymes can cleave the PAR N-terminus, but the endogenous protease activators of PARs in OA remain unclear. Here we characterized PAR activating enzymes in knee joint synovial fluids from OA patients and healthy donors using genetically encoded PAR biosensor expressing cells. Calcium signaling assays were performed to examine receptor activation. The class and type of enzymes cleaving the PARs was further characterized using protease inhibitors and fluorogenic substrates. We find that PAR1, PAR2 and PAR4 activating enzymes are present in knee joint synovial fluids from healthy controls and OA patients. Compared to healthy controls, PAR1 activating enzymes are elevated in OA synovial fluids while PAR4 activating enzyme levels are decreased. Using enzyme class and type selective inhibitors and fluorogenic substrates we find that multiple PAR activating enzymes are present in OA joint fluids and identify serine proteinases (thrombin and trypsin-like) and matrix metalloproteinases as the major classes of PAR activating enzymes in the OA synovial fluids. Synovial fluid driven increase in calcium signaling was significantly reduced in cells treated with PAR1 and PAR2 antagonists, but not in PAR4 antagonist treated cells. OA associated elevation of PAR1 cleavage suggests that targeting this receptor may be beneficial in the treatment of OA.


Materials and methods
All studies with human samples were approved by the Western University Health Sciences Research Ethics Board and were in accordance with the Declaration of Helsinki. All research complied with relevant guidelines and informed consent was obtained from all participants. Healthy donor knee synovial fluids were obtained from Rheumatology Centre (C.T.A) at St. Joseph's Health Care London, ON, Canada (ethics ID # REB 109255). Synovial fluid samples from OA patients undergoing realignment osteotomy were obtained from the Fowler Kennedy Sport Medicine Clinic and University Hospital at London Health Sciences Centre, London, ON, Canada (ethics ID # REB 108039). At surgery, OA patient synovial fluids from knee joints were collected by an orthopedic surgeon (A.G or R.B.L) prior to commencing arthroscopy and proximal tibial osteotomy. All samples were immediately placed on ice and transferred to the laboratory, centrifuged at 10,000 × g for 1 min, aliquoted and frozen at − 80 °C. All joint fluids were tested for presence of any blood with the Fecal Occult Blood Test kit (detection level: 0.6 mg Hb/g, Immunostics Hema-Screen) and samples showing presence of blood were excluded from further analysis.
Thrombin (human plasma, specific activity ≥ 2800 NIH units/mg protein, Calbiochem-EMD Millipore) and trypsin (porcine pancreas, Type IX-S, 13,000-20,000 BAEE units/mg protein, Sigma-Aldrich) stock solutions were made in 25 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, Fisher Scientific). The thrombin-selective inhibitor PPACK.2HCl and MMP Inhibitor V (ONO-4817) were obtained from Calbiochem (Millipore Sigma). The broad-spectrum MMP inhibitor batimastat (BB-94, ≥ 98%) was from Sigma-Aldrich. Soybean trypsin inhibitor (STI) was from ThermoFisher Scientific or Millipore Sigma. The fluorogenic substrates, Bz-Phe-Val-Arg-AMC.HCl (Thrombin substrate III) and Boc-Gln-Ala-Arg-AMC.HCl (Trypsin substrate) were from Bachem, Suc-Ala-Ala-Pro-Phe-AMC (Chymotrypsin substrate II) and Z-Gly-Gly-Arg-AMC.HCl (Urokinase substrate III) were from Calbiochem, and MCA-Lys-Pro-Leu-Gly-Leu-Dpa(DNP)-Ala-Arg-NH 2 (MMP substrate FS-6) was from Sigma-Aldrich. The stock solutions of the enzyme inhibitors and fluorogenic substrates were prepared according to manufacturer's instructions. PAR agonist peptides TFLLR-NH 2 (PAR1), SLIGRL-NH 2 (PAR2) and AYPGKF-NH 2 (PAR4) were from GenScript Biotech, and stock solutions were made in 25 mM HEPES. PAR1 selective antagonist Vorapaxar was purchased from Adooq Bioscience, PAR2 antagonist AZ3451 from Sigma, and PAR4 antagonist BMS-986120 from Cayman Chemical. The stock solutions of the PAR antagonists were prepared in dimethyl sulfoxide (DMSO, BioShop). All samples were diluted to appropriate working concentrations in Hanks' Balanced Salt Solution containing CaCl 2 and MgCl 2 (HBSS, Gibco ThermoFisher Scientific). Fluorogenic substrates were diluted in Tris-NP40-calcium buffer [ www.nature.com/scientificreports/ Patient inclusion criteria. Patients with a primary diagnosis of symptomatic knee OA undergoing a coronal plane alignment correction by opening wedge proximal tibial osteotomy were included. Patients undergoing concomitant procedures such as ligament reconstruction, meniscal transplantation or cartilage restoration were excluded. Patients with inflammatory arthropathy or past history of joint infection were also excluded. All patients completed a radiological examination including weight bearing anteroposterior, 45-degree flexed posteroanterior, lateral and standing hip-knee-ankle alignment views. The amount of radiographic knee OA was graded independently by two orthopedic surgical fellows on a scale of grade 0 (none), 1 (doubtful), 2 (mild), 3 (moderate), and 4 (severe), according to the Kellgren and Lawrence (KL) classification system 47 . Absence of knee symptoms and radiographically confirmed KL 0 was considered healthy. Patients also completed Knee Injury and Osteoarthritis Outcome Score (KOOS) 48 , the Western Ontario and McMaster Universities Arthritis Index (WOMAC) 49 , and Visual Analog Scale (VAS) pain score 50 questionnaires at baseline prior to surgery. The total KOOS is a mean percentage score of the five subscales encompassing pain, symptoms, activities in daily living function (ADL), sport and recreation function (Sport/Rec), and quality of life (QOL), score mean 48,51 . The total WOMAC is a mean percentage score of the three subscales encompassing pain, stiffness, and physical function 49 .
In addition, each subscale score was calculated independently and transformed to a percentage score. Each score was converted to a percentage score by using the formula below, where KOOS and WOMAC transformed score of 100% represents no problems and 0% indicates extreme problems. VAS pain score rates the patient's pain at rest and during activity (move) on average over the past week, and the scale ranges from 0 (no pain) to 10 (worst pain possible). The mark placed along the scale (0-10 cm) by the patient is measured as the level of pain for each situation 50 .
Biosensor cloning and stable transfection. Human PAR2 with N-terminal nano-luciferase (nLuc, Promega) and C-terminal enhanced Yellow Fluorescent Protein (eYFP) tagged constructs have been previously described 53 . N-Terminal nLuc tagged human PAR1 and PAR4 constructs were similarly constructed by generating restriction enzyme sites for BspE1 and BamH1 by site-directed mutagenesis (QuickChange, Agilent technologies) and inserting nLuc in previously described eYFP tagged receptor constructs 38,52 . nLuc was located between the residues Glu 30 Ser 31 in PAR1, Gly 28 Thr 29 in PAR2, and Pro 23 Ser 24 in PAR4. Fidelity of all constructs was verified by direct sequencing (London Regional Genomics Centre, Robarts Research Institute). CHO-K1 cells were stably transfected with the nLuc and eYFP tagged hPAR1, hPAR2, or hPAR4-pcDNA3.1( +) constructs by electroporation (Super Electroporator NEPA21 Type II, Nepa Gene) with 3 µg plasmid DNA and 1 × 10 6 cells in 100 µl Opti-MEM (1 ×) Reduced Serum Medium (Gibco ThermoFisher Scientific). The electroporated cells were cultured in a non-selective complete medium in a 100 mm × 20 mm cell culture dish (Falcon, Corning) for 48-72 h. The cells were subsequently maintained in G418 selective medium in a T75 flask for 7-14 days. G418-resistant cells expressing the construct (nLuc-hPAR1/2/4-eYFP) were clonally sorted by flow cytometry (FACSAriaIII, London Regional Flow Cytometry Facility, Robarts Research Institute) and expanded in G418 selective medium. The stably transfected reporter cell lines were characterized with a known hPAR agonist, thrombin on nLuc-hPAR1-eYFP, trypsin on nLuc-hPAR2-eYFP, and both thrombin and trypsin on nLuc-hPAR4-eYFP, using the luciferase assay technique described below. As a negative control, luminescence levels in the parental CHO-K1 cells treated with PAR agonists thrombin and trypsin were also assessed.
Luciferase assay. The presence of active PAR cleaving enzymes was measured by monitoring release of the N-terminal nLuc fusion tag. The nLuc-hPAR1/2/4-eYFP-CHO reporter cells were plated either in a 24-well or 96-well cell culture plates (polystyrene, flat-clear bottom, Nunclon Delta, Nunc, ThermoFisher Scientific) at a cell density of 1 × 10 4 cells per well in complete F-12 medium and cultured for 48 h. The cells were rinsed with HBSS (3 × 100 µl) and incubated with 100 µl HBSS at 37 °C for 15 min. 50 µl of cell supernatant from each well was transferred into a 96-microwell white plate (polystyrene, Nunclon Delta, Nunc, ThermoFisher Scientific) and this served as a measure of basal luminescence levels in each well. The cells were then incubated with 50 µl of test samples, recombinant enzymes or controls at 37 °C for 15 min. 50 µl of cell supernatant from treated wells were transferred to a white plate. The Nano-Glo Luciferase Assay Substrate furimazine (2 µl ml −1 , Promega) was Transformed score = 100% − actual raw score maximum score × 100% . Calcium signaling assay. Synovial fluids activation of PAR mediated calcium signaling was assessed in HEK293 cells that endogenously express PAR1 and PAR2, and stably transfected PAR4. PAR activated Gα q/11mediated calcium signaling was measured as described previously 54  Mann-Whitney U test with 95% confidence intervals (CI) was utilized to compare any statistical significance between two groups, and one-way ANOVA Kruskal-Wallis test was utilized to assess differences between multiple groups, at p < 0.05. Cohen's kappa (κ) was used to determine the inter-rater agreement between the two clinicians when grading KL knee OA. Agreement was interpreted using the scale κ < 0.20: slight agreement, κ = 0.21-0.40: fair agreement, κ = 0.41-0.60: moderate agreement, κ = 0.61-0.80: substantial agreement, and κ = 0.81-1.0: almost perfect agreement 55 . Pearson correlation coefficient analysis on GraphPad Prism 8 was used to determine the correlation between PAR activity and subscales of KOOS/WOMAC/VAS scores. Correlation coefficient was interpreted based on the reported scale 56 r = ± 1.00 to ± 0.90: a very strong correlation, r = ± 0.89 to ± 0.70: a strong correlation, r = ± 0.69 to ± 0.40: a moderate correlation, r = ± 0.39 to ± 0.10: a weak correlation, and r = ± 0.10 to ± 0.00: negligible correlation.

Results
Patient characteristics. Demographics and radiographic data of the four healthy donors (H1-H4) and twenty-five OA patients  are presented in Table 1. The degree of radiographic knee OA was graded on a scale of grade 0 (none), 1 (doubtful), 2 (mild), 3 (moderate) and 4 (severe) according to the KL classification system 47

PAR1, PAR2 and PAR4 cleavage by enzymes in synovial fluids.
Cleavage of PAR1, PAR2 and PAR4 was assessed with the four healthy (H1-H4) and twenty-five OA patients' (1-25) synovial fluids (10%) using the nLuc-hPAR1/2/4-eYFP-CHO reporter cell lines (Fig. 2). A significantly higher level of PAR1 cleavage was detected in OA patient synovial fluids treated cells compared to the healthy fluid treated cells ( Fig. 2A). Interestingly, cleavage of PAR2 was not significantly different between healthy and OA synovial fluids (Fig. 2B), while cleavage of PAR4 was significantly lower (Fig. 2C) with OA patient knee joint fluids compared to the healthy control samples. It must be noted that these assays were done with a 1:10 dilution of the knee joint fluids and indicated cleavage of ~ 10-20% of maximum thrombin (3 U ml −1 ) activity in PAR1 and ~ 15-20% of maximum trypsin (100 nM) activity in PAR2, respectively. This would translate to the presence of ~ 3-6 U ml −1 of thrombin-like enzymatic activity and 15-20 nM trypsin-like enzymatic activity in the knee joint fluids. Sample sizes were not large enough in this study to correlate enzyme activity levels with radiological OA severity.
To examine if PAR cleavage activity in synovial fluids is influenced by patient characteristics, we stratified the PAR1, PAR2, and PAR4 cleavage data further based on patient demographics including sex, BMI, and age ( Figure S1). Interestingly, PAR1 ( Figure S1-A(i)) and PAR2 ( Figure S1-B(i)) cleavage was higher with synovial fluids from males compared to females, while PAR4 cleavage was higher in females relative to males ( Figure  S1-C(i)). There were no differences seen in PAR1, PAR2, or PAR4 cleavage activity between synovial fluids from normal weight (BMI 18.5-24.9 kg m −2 ), overweight (BMI 25.0-29.9 kg m −2 ), and obese (BMI > 29.9 kg m −2 ) patients ( Figure S1-(ii)) as well as across different age groups ( Figure S1-(iii)). Given the relatively small sample size in different categories, the trends observed here are interesting but need to be confirmed in a larger patient cohort in future studies.
To further clarify the nature of different enzymes present in the synovial fluids, we used peptide-based fluorogenic substrates of thrombin, trypsin, chymotrypsin, urokinase, and MMPs. With the use of thrombin substrate Bz-Phe-Val-Arg-AMC ( Figure S2-A), trypsin and matriptase substrate Boc-Gln-Ala-Arg-AMC ( Figure  S2-B), and a broad-spectrum MMP substrate MCA-Lys-Pro-Leu-Gly-Leu-Dpa(DNP)-Ala-Arg-NH 2 which can be hydrolyzed by MMP-1, 2, 3,7,8,9,12,13,14,16,20, ADAM-10, 17/TACE and BACE2 75-78 , significant levels of thrombin-like enzymes (Fig. 4A), trypsin-like enzymes (Fig. 4B) and MMPs (Fig. 4C) activity were detected in the twenty-five patients' synovial fluids (10%) tested. However, no cleavage of the chymotrypsin sensitive substrate Suc-Ala-Ala-Pro-Phe-AMC, or the urokinase and plasminogen activators sensitive substrates Z-Gly-Gly-Arg-AMC.HCl was detected with any of the twenty-five patient synovial fluids (data not shown). Remarkably, thrombin-like and trypsin-like enzymes were higher in OA samples compared to healthy synovial fluids (Fig. 4A,  B), which was consistent with the high PAR1 cleavage found with the OA patient samples in the PAR biosensor assay (Fig. 2A). The limited volume of synovial fluids available from healthy volunteers prevented assessment of healthy samples with the MMP substrate.
As observed in the PAR biosensor cleavage assay (Fig. 3), significant inhibition of thrombin-like (Fig. 4A), trypsin-like (Fig. 4B) and MMP (Fig. 4C) enzymes activity was found in all the OA patient synovial fluids pretreated with PPACK (1 µM), STI (1 mg ml −1 ) or BB-94 (10 µM), respectively. In addition, here we used another MMP inhibitor ONO-4817 and as observed with BB-94, a significant reduction in MMP activity was seen in the ONO-4817 (10 µM) pretreated patients' synovial fluids (Fig. 4C). This suggests that one or more MMP is present in OA synovial fluids. Representative enzymatic activity kinetic curves are shown in Fig. 4D-F. Together, this set of experiments further confirmed the presence of significant levels of serine proteinases and matrix metalloproteinases in the OA knee joint fluids.
The substrate cleavage enzyme activity data was also analyzed in the context of the OA patient demographics ( Figure S3). However, there was no substantial difference seen in enzyme activity by sex of the patients. This contrasts with the observations seen with the PAR biosensor cleavage assay data ( Figure S1-(i)). As seen with PAR cleavage, enzyme activity did not show a significant difference between normal weight, overweight, or obese patients. It was however interesting to see a significantly lower level of thrombin-like enzyme activity in older adults relative to middle-aged adults ( Figure S3-A(iii)). There was no significant difference seen in the trypsin-like enzyme activity between different age groups ( Figure S3-B(iii)) in agreement with observations with the PAR2 cleavage data ( Figure S1-B(iii)). Interestingly, MMP activity was significantly higher in older adults compared to young or middle-aged patients ( Figure S3-C(iii)). Together, there seems to be an interesting link between OA patient demographics, enzyme activity, and PAR cleavage. The sample size in this human pilot study was however not large enough to make a strong statistical comparison and these observations needs to be confirmed in a larger patient cohort in the future. One-Way ANOVA Kruskal-Wallis test was utilized to assess differences between groups. p < 0.05 was considered statistically significant. It is well established that PAR activating enzymes, thrombin (PAR1 and PAR4) and trypsin (PAR2), and PAR specific agonist peptides, TFLLR-NH 2 (PAR1), SLIGRL-NH 2 (PAR2) and AYPGKF-NH 2 (PAR4) trigger robust calcium release ( Figure S4-A) that is dependent on Gα q/11 -coupled signaling pathway 52,79-81 . PAR mediated calcium signaling can be inhibited using the antagonists, Vorapaxar (PAR1), AZ3451 (PAR2) and BMS-986120 (PAR4) 79 , in a concentration-dependent manner ( Figure S4-B). OA synovial fluids (10%) triggered an increase in calcium signaling in HEK293 cells expressing PAR1, PAR2 and PAR4 (Figs. 5 and S5). Pre-treatment of cells with Vorapaxar (PAR1) and AZ3451 (PAR2) resulted in a significant drop in calcium signaling (Figs. 5 and S5) consistent with our PAR cleavage data where PAR1 and PAR2 cleavage was monitored ( Fig. 2A, B). Curiously, a significant increase in intracellular calcium was seen in cells that had been treated with the PAR4 antagonist BMS-986120 prior to OA synovial fluid treatment (Figs. 5 and S5-B).

Correlation of KOOS, WOMAC, and VAS scores to PAR1/2/4 activity. OA patients self-reported
arthritic questionnaires scores including Knee Injury and Osteoarthritis Outcome Score (KOOS) 48 , the Western Ontario and McMaster Universities Arthritis Index (WOMAC) 49 and Visual Analog Scale (VAS) pain score 50 at baseline prior to surgery (Table S1) were correlated to the obtained PAR1, PAR2, and PAR4 cleavage data to identify if any correlation exists ( Figure S6)  Figure S6). Overall, the sample sizes in the current study are not sufficient to do robust correlation analysis however such analysis of larger patient cohorts is likely to be informative.

Discussion
Using novel biosensor expressing reporter cells and fluorogenic substrates we have monitored PAR cleavage and proteolytic activity in knee joint fluids from twenty-five OA patients with disease severity ranging from KL grade 1-3. We find multiple active PAR1, PAR2, and PAR4 activating enzymes, including serine proteinases and metalloproteinases, in human knee joint synovial fluids tested. Curiously, levels of PAR1 activating enzymes were elevated and PAR4 activating enzyme levels decreased in the OA joint fluids but not in the healthy joint fluids studied here. This decline in PAR4 activating enzymes in OA joint fluids is interesting since some PAR1 and PAR2 activating enzymes, including thrombin and trypsin, also activate PAR4. This divergent activation of PAR1 and PAR4 in OA suggests that there are distinct PAR1 and PAR4 activating enzymes in the knee joint that should be identified in order to better understand the roles of these receptors in OA pathogenesis. Proteolytic enzymes are important mediators of joint health and pathophysiology. The role of MMPs in particular, as a class of enzymes that digest ECM components and cause cartilage degradation, is well established 82 . More recently it has emerged that other classes of enzymes such as serine proteinases are also upregulated in OA and may participate in proteolytic cascades leading to cartilage destruction 83 . Many of the enzymes that are implicated in OA pathology also trigger pro-inflammatory signaling cascades through the activation of the PAR family of GPCRs 8 .
PARs have been studied in the context of arthritis using several animal models. PAR1 deficient mice showed decreased cartilage degradation, and lower levels of synovial cytokine mRNA and MMP-13 mRNA in a model of antigen-induced arthritis 28 . PAR1 deficiency is also protective in a model of psoriatic arthritis driven by increased dermal expression of kallikrein 6 84 . In contrast, PAR1 deficiency did not afford significant protection in the destabilization of the medial meniscus (DMM) model of OA 27 . PAR2 deletion or pharmacological inhibition on the other hand has shown more consistent protective effects across multiple models, though there are some variations in the degree of reduction in cartilage erosion and subchondral bone thickening reported 14,17,27 . The role of PAR4 expressed in joint cells or tissues is not as well studied, however PAR4 activation significantly inhibits PAR2 agonist and transient receptor potential vanilloid-4 (TRPV4) agonist-mediated visceral pain 85 and PAR4 activation can also decrease excitability in dorsal root ganglion neurons 86 . PAR4 signaling may therefore be protective in the context of osteoarthritis pain. Overall, the disparate results across different models point to PAR signaling in different immune and joint cells contributing to the disease, with PAR expression well established in chondrocytes, fibroblast-like synoviocytes, osteoblasts, immune cells, and nociceptors 8 . Further studies with tissue-specific deletion of PARs across multiple models of arthritis as well as careful analysis of possible species differences are required to fully understand the relative contributions of each receptor and cell type in human OA.
Several proteolytic enzyme activators of PARs are nonetheless implicated in OA and similar protection of joints in OA is also reported when the key PAR1 and PAR2 activating enzymes are inhibited. Serine proteinases involved in the coagulation and fibrinolysis cascades, including thrombin, plasminogen activators and plasmin, are well established as activators of PAR1 and show a substantial increase in the inflamed joint of OA patients and animal models 87,88 . MMPs including MMP-1, 2, 3, 9, 13 and 14 are secreted in response to inflammatory cytokines and growth factors by chondrocytes and synoviocytes 4,82 . A number of these MMPs including MMP-1, 2, 3, 8, 9, 12 and 13 are able to activate PARs. In addition, immune cell-derived proteinases such as mast cell Figure 5. OA joint fluid activation of PAR mediated calcium signaling. Synovial fluids (10%) were added to HEK293 cells expressing PAR1, PAR2 and PAR4. Elevation in intracellular calcium levels were monitored in (A) untreated cells and cells pretreated with PAR antagonists, Vorapaxar, 1000 nM (PAR1 antagonist), AZ3451, 1000 nM (PAR2 antagonist) and BMS986120, 1000 nM (PAR4 antagonist). Scatter plots depict peak calcium signaling levels in individual experiments as a percentage of the response elicited by the synovial fluid samples in untreated cells. One-Way ANOVA Kruskal-Wallis test was utilized to assess differences between groups. p < 0.05 was considered statistically significant. (B) A representative calcium signaling trace obtained for PAR mediated calcium signaling triggered by one patient sample in the absence and presence of PAR1, PAR2, and PAR4 antagonists. The data represent the mean ± SEM of three independent experiments (N = 3) performed in duplicate.  38 and cathepsin-S (Gly 40 /Lys 41 ) 39 also cleave the receptor at different sites than trypsin. Each of these different cleavage events can result in different signaling responses, some of which may be protective. While we show that PARs can be cleaved by various enzymes in OA joint fluids, a thorough characterization of receptor coupling to different G protein and β-arrestin mediated signaling pathways is necessary to fully understand the role of this signaling system in joint health and pathology. The diversity of PAR activators in the joints also highlight the importance of understanding signaling differences elicited by the different PAR activating enzymes in testing appropriate therapeutic interventions. Interestingly, the significant reduction in OA joint fluid triggered calcium signaling in the presence of a PAR1 antagonist along with the elevation of PAR1 cleaving enzyme activity in OA synovial fluids suggests that inhibition of PAR1 could be a promising therapeutic strategy to explore in OA. On the other hand, inhibition of PAR4 led to an increase in calcium signaling indicating that PAR4 mediated signaling may be producing factors or signaling events that inhibit responses from PAR1 and PAR2. Given the significant decrease in PAR4 activating enzyme levels in OA patients, this suggests the intriguing hypothesis that PAR4 signaling is a protective response in the knee joint that is lost in OA. While a significant difference between healthy and OA synovial fluid PAR2 activating enzymes was not noted in our study, PAR2 activating enzymes are nevertheless present in the knee joint synovial fluid. PAR2 is known to be an important regulator of immune cell infiltration and may contribute to OA pathogenesis. Further analysis of samples from different types and severity of OA are required to fully correlate PAR activating protease levels with disease pathogenesis.

Scientific
In summary, we have established and characterized here a novel biosensor expressing cell line and an assay that allows rapid and facile screening of PAR cleaving enzymes in complex biological fluids. In a small population of OA patients, we were able to detect multiple enzymes that target PARs and find elevated levels of PAR1 cleaving enzymes in OA. We further verified that PAR cleavage led to increases in receptor mediated calcium signaling that could be inhibited using PAR1 and PAR2 specific antagonists. This study broadly defines proteinase classes that are present in the arthritic joint and guide future studies aimed at isolating and further characterizing endogenous regulators of PAR receptors in the healthy and diseased joints.

Data availability
All data generated or analysed during this study are included in this published article and its Supplementary Information file. www.nature.com/scientificreports/